Dicarboxylic acid production by fermentation at low pH

ABSTRACT

The present invention relates to a process for the production of a dicarboxylic acid. The process comprises fermenting a yeast in the presence of a carbohydrate-containing substrate and low amounts of oxygen at a pH value at which at least 50% of the dicarboxylic acid is in the acid form. The process of the present invention allows for high yields of the dicarboxylic acid product and is more cost-effective than existing processes in which the salt is produced which during recovery has to be converted to the acid. It also leads to a simpler and more convenient downstream processing.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/003,217, filed Jan. 7, 2011, which is a 371 application ofPCT/EP09/56181, filed May 20, 2009, which claims priority from EP08159891.4, filed Jul. 8, 2008, the contents of which are incorporatedherein by reference in their entireties.

BACKGROUND Field of the invention

The present invention relates to a process for the production ofdicarboxylic acids. In particular, it relates to the production ofdicarboxylic acids by fermentation of a yeast.

Dicarboxylic acids, such as fumaric acid and succinic acid, areimportant compounds which are used in the food industry for thepreparation and preservation of food, in the medical industry for theformulation of medical products and other industrial uses, such asmonomers for (bio)polymers. To meet the increasing need for dicarboxylicacids, more efficient and cost effective production methods are beingdeveloped. Traditionally, dicarboxylic acids are made by fermentation ofbacteria, which can produce large amounts of dicarboxylic acids. This isfor example described in U.S. Pat. No. 5,573,931 which describes amethod for producing succinic acid in high concentrations by employing abacterial strain. However, one major drawback associated with the use ofbacteria for producing dicarboxylic acids is the formation ofdicarboxylic acid salt. If bacteria are used, the pH during fermentationneeds to be maintained in the range of pH 6-7, which is higher than thepKa values of all dicarboxylic acids. As a consequence, most acids willbe produced in their salt form and the salts will have to be convertedinto the acid. This is not practical or efficient in large-scaleproduction processes and raises production costs.

Also microorganisms other than bacteria have been employed for theproduction of organic acids. EP 0 424 384 discloses an aerobic processfor the production of organic acids by Rhizopus in a medium containingcalcium carbonate. EP 1 183 385 discloses genetically manipulated yeastcells with a Crabtree negative phenotype and containing an exogenousnucleus acid molecule for the production of lactic acid.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The present invention relates to a process for the production of adicarboxylic acid. The process comprises fermenting a yeast in thepresence of a carbohydrate-containing substrate and low amounts ofoxygen at a pH value which is below the pKa of the dicarboxylic acid.The process of the present invention allows for high yields of thedicarboxylic acid product, allows for a simpler downstream processingand is more cost-effective than existing processes in which the salt isproduced which has then to be converted to the acid. Since dicarboxylicacids have more than one pKa value, the pH should be below the lowestpKa of the dicarboxylic acid. For most acids, the pH will typically bein the range of pH 1.0 to pH 5.5, preferably between pH 2.0 and pH 4.0.In one embodiment, succinic acid is produced at a pH value of 3.0.Another advantage is that due to the low pH the risk of contamination isreduced.

The acid production phase is preferably preceded by a biomass formationphase for optimal biomass production. In the biomass formation phase thepH is in the range of pH 2 to pH 7. Preferably, the pH is in the rangeof pH 3 to pH 6, more preferably, the pH is in the range of pH 4 to pH5.

The process according to the present invention is more cost-effectiveand may lead to a 30% lower cost price. One of the reasons is thattitrant costs are significantly reduced.

The process may be used for the production of any dicarboxylic acid.Suitable examples include adipic acid, fumaric acid, itaconic acid,succinic acid, malic acid, oxalic acid. Preferably, the dicarboxylicacid is succinic acid, fumaric acid or malic acid.

The yeast which is used in the process may be any suitable yeast.Suitable examples of yeasts include Saccharomyces, Schizosaccharomyces,Kluyveromyces, Candida, Pichia and Yarrowia, such as species ofSaccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyvermoceslactis, Candida sonorensis, Pichia stipidis and Yarrowia lipolytica. Inone embodiment, the eukaryotic microorganism used in the process is aSaccharomyces cerevisiae, a microorganism which is a widely usedindustrially interesting microorganism.

In a preferred embodiment the yeast according to the present inventionis a genetically modified yeast. As used herein, a genetically modifiedyeast in the process according to the present invention is defined as ayeast cell which contains, or is transformed or genetically modifiedwith a nucleotide sequence or polypeptide that does not naturally occurin the yeast cell, or it contains additional copy or copies of anendogenous nucleic acid sequence. A wild-type yeast cell is hereindefined as the parental cell of the recombinant cell.

Preferably, the yeast in the process according to the present inventionis a genetically modified yeast comprising a nucleotide sequenceencoding a heterologous enzyme selected from the group consisting of aphosphoenolpyruvate carboxykinase, fumarate reductase and a fumarase.Preferred embodiments of the heterologous enzymes are as defined hereinbelow.

The term “homologous” when used to indicate the relation between a given(recombinant) nucleic acid or polypeptide molecule and a given hostorganism or host cell, is understood to mean that in nature the nucleicacid or polypeptide molecule is produced by a host cell or organisms ofthe same species, preferably of the same variety or strain.

The term “heterologous” when used with respect to a nucleic acid (DNA orRNA) or protein refers to a nucleic acid or protein that does not occurnaturally as part of the organism, cell, genome or DNA or RNA sequencein which it is present, or that is found in a cell or location orlocations in the genome or DNA or RNA sequence that differ from that inwhich it is found in nature. Heterologous nucleic acids or proteins arenot endogenous to the cell into which it is introduced, but have beenobtained from another cell or synthetically or recombinantly produced.

Preferably the genetically modified yeast comprises a nucleotidesequence encoding a phosphoenolpyruvate carboxykinase. The PEPcarboxykinase (EC 4.1.1.49) preferably is a heterologous enzyme,preferably derived from bacteria, more preferably the enzyme having PEPcarboxykinase activity is derived from Escherichia coli, Mannheimia sp.,Actinobacillus sp., or Anaerobiospirillum sp., more preferablyMannheimia succiniciproducens or Actinobacillus succinogenes. In oneembodiment the PEP carboxykinase is derived from Actinobacillussuccinogenes (PCKa), wherein the PCKa preferably has been modified toreplace EGY at position 120-122 with a DAF amino acid sequence.Preferably, a yeast cell according to the present invention isgenetically modified with a PEP carboxykinase which has at least 80, 85,90, 95, 99 or 100% sequence identity with amino acid sequence of SEQ IDNO: 6.

In another preferred embodiment a genetically modified yeast in theprocess according to the present invention comprises a nucleotidesequence encoding a fumarate reductase. Preferably, the fumaratereductase is a heterologous enzyme, preferably a NAD(H)-dependentfumarate reductase, which may be derived from any suitable origin, forinstance bacteria, fungi, protozoa or plants. Preferably, a yeast in theprocess according to the invention comprises a heterologousNAD(H)-dependent fumarate reductase, preferably derived from aTrypanosoma sp., for instance a Trypanosoma brucei. In a preferredembodiment the nucleotide sequence encoding a NAD(H)-dependent fumaratereductase is expressed in the cytosol. In the event that the nucleotidesequence encoding a NAD(H)-dependent fumarate reductase comprises aperoxisomal or mitochondrial targeting signal, it may be essential tomodify or delete a number of amino acids (and corresponding nucleotidesequences in the encoding nucleotide sequence) in order to preventperoxisomal or mitochondrial targeting of the enzyme. The presence of aperoxisomal targeting signal may for instance be determined by themethod disclosed by Schlöuter et at, Nucleic acid Research 2007, 35,D815-D822. Preferably, a yeast cell according to the present inventionis genetically modified with a NAD(H)-dependent fumarate reductase,which has at least 80, 85, 90, 95, 99 or 100% sequence identity with SEQID NO: 7.

In another preferred embodiment a genetically modified yeast in theprocess according to the present invention comprises a nucleotidesequence encoding a fumarase, which may be a heterologous or homologousenzyme. A nucleotide sequence encoding a heterologous fumarase may bederived from any suitable origin, preferably from microbial origin,preferably from a yeast, for instance Saccharomyces cerevisiae or afilamentous fungus, for instance Rhizopus oryzae. Preferably, a yeast inthe process according to the present invention overexpresses anucleotide sequence encoding a fumarase that has at least 70%,preferably at least 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, or 99% or100% sequence identity with the amino acid sequence of SEQ ID NO: 8.

In another preferred embodiment a genetically modified yeast in theprocess according to the present invention further comprises anucleotide sequence encoding a malate dehydrogenase (MDH) which isactive in the cytosol upon expression of the nucleotide sequence.Preferably, the MDH lacks a peroxisomal or mitochondrial targetingsignal in order to localize the enzyme in the cytosol. A cytosolic MDHmay be any suitable homologous or heterologous malate dehydrogenase.Preferably, a yeast cell according to the present invention comprises anucleotide sequence encoding a malate dehydrogenase that has at least70%, preferably at least 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, 99%sequence identity with the amino acid sequence of SEQ ID NO: 9.

In another embodiment, a genetically modified yeast in the processaccording to the invention comprises a nucleotide sequence encoding adicarboxylic acid transporter protein, preferably a malic acidtransporter protein (MAE). A dicarboxylic acid transporter protein maybe a homologous or heterologous protein. Preferably the dicarboxylicacid transporter protein is a heterologous protein. A dicarboxylic acidtransporter protein may be derived from any suitable organism,preferably from Schizosaccharomyces pombe. Preferably, a dicarboxylicacid transporter protein is a malic acid transporter protein (MAE) whichhas at least 80, 85, 90, 95 or 99% or 100% sequence identity with SEQ IDNO: 10.

Preferably, the yeast used in the process according to the presentinvention is a genetically modified yeast comprising a heterologousPEP-carboxykinase, a heterologous NAD(P)H-dependent fumarate reductase,a heterologous fumarase, a heterologous malic acid transporter proteinand a cytosolic malate dehydrogenase. Preferred embodiments of theseenzymes are as defined herein above.

Sequence identity is herein defined as a relationship between two ormore amino acid (polypeptide or protein) sequences or two or morenucleic acid (polynucleotide) sequences, as determined by comparing thesequences. Usually, sequence identities or similarities are comparedover the whole length of the sequences compared. In the art, “identity”also means the degree of sequence relatedness between amino acid ornucleic acid sequences, as the case may be, as determined by the matchbetween strings of such sequences.

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include BLASTP and BLASTN, publicly available fromNCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIHBethesda, Md. 20894). Preferred parameters for amino acid sequencescomparison using BLASTP are gap open 11.0, gap extend 1, Blosum 62matrix.

The term “nucleic acid” as used herein, includes reference to adeoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, ineither single-or double-stranded form, and unless otherwise limited,encompasses known analogues having the essential nature of naturalnucleotides in that they hybridize to single-stranded nucleic acids in amanner similar to naturally occurring nucleotides (e.g., peptide nucleicacids). A polynucleotide can be full-length or a subsequence of a nativeor heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof.

In a preferred embodiment, the yeast in the process according to theinvention overexpresses the nucleotide sequences encoding any of theenzymes as defined herein above. There are various means available inthe art for overexpression of nucleotide sequences encoding enzymes in ayeast in the process of the invention. In particular, a nucleotidesequence encoding an enzyme may be overexpressed by increasing the copynumber of the gene coding for the enzyme in the cell, e.g. byintegrating additional copies of the gene in the cell's genome, byexpressing the gene from a centromeric vector, from an episomalmulticopy expression vector or by introducing an (episomal) expressionvector that comprises multiple copies of the gene. Preferably,overexpression of the enzyme according to the invention is achieved witha (strong) constitutive promoter.

The carbohydrate-containing substrate may be any carbohydrate containingsubstrate including molasse, sugar cane juice, pentoses and hexoses,such as glucose, fructose, xylose, arabinose. Preferably, thecarbohydrate-containing substrate is a glucose-containing substrate,such as maltose, sucrose, glucose or a glucose syrup. The carbohydratecontent of the carbohydrate-containing substrate is preferably more than50% w/w, more preferably more than 55%, 60%, 65%, 70%, 75%, 80% w/w,most preferably more than 85%, 90%, 95% or 99% w/w on the basis of drymatter content.

The process according to the present invention, preferably comprisesfermenting a yeast under carbon (C)-limited conditions. C-limitedconditions are defined herein as a concentration of dissolvedcarbohydrate of below 1 g/l, preferably below 0.9 g/l, 0.8 or below 0.5g/l of dissolved carbohydrate. It was found that fermenting yeast underC-limited conditions resulted in an increased yield of succinic acid ascompared to non-C-limited conditions.

The oxygen for the fermentation may be supplied in any suitable form. Inone embodiment, the oxygen is supplied in the form of air. The oxygenshould be supplied in low amounts. This is reflected in the oxygenuptake rate (OUR) and/or the specific oxygen uptake rate (qO₂) of theyeast. The OUR in the present invention is lower than about 8.0 mmoloxygen/L/hour, preferably lower than about 5.0, 4.0, 3.0, or 2.0 mmoloxygen/L/hour, more preferably lower than about 1.0, or 0.5 mmoloxygen/L/hour, preferably above 0.01 mmol oxygen/L/hour.

The specific oxygen uptake rate (qO₂) in the process of the inventionranges between 8 mmol oxygen/g biomass dry weight/hour to 0.5 mmoloxygen/g biomass dry weight/hour, preferably between 5, 4, 3, or 2 mmoloxygen/g biomass/hour to about 0.4, 0.3, or 0.2 mmol/oxygen/gbiomass/hour.

The process according to the present invention may be carried out inbatch, fed-batch or continuous mode. These fermentation modes are knownto the skilled man in the art. Depending on the fermentation mode, thebiomass concentration during fermentation may vary more or less duringfermentation. In batch and fed-batch mode the biomass concentrationusually increases. Consequently, the specific oxygen uptake rate usuallydecreases in a batch and fed-batch mode.

The temperature of the process is typically between 10 and 40 degreesC., preferably between 20 and 35 degrees C., more preferably between 30and 35 degrees C.

In one embodiment of the process according to the invention, an extraelectron donor is present in addition to the carbohydrate-containingsubstrate. The extra electron donor is preferably an organic electrondonor. Suitable examples of organic electron donors include glycerol,formate and polyols, such as mannitol, sorbitol and xylitol.

FIGURES

FIG. 1. Effect of the applied OUR on the succinic acid production after90 h at pH 3.

EXAMPLES Example 1 Succinic Acid Production by Saccharomyces cerevisiae

1.1. Construction Yeast Strain

1.1.1. Construction of Expression Constructs

Expression construct pGBS414PPK-3 was created after a BamHI/NotIrestriction of the S. cerevisiae expression vector pRS414 (Sirkoski R.S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligatingin this vector a BamHI/NotI restriction fragment consisting of thephosphoenolpyruvate carboxykinase (origin Actinobacillus succinogenes)synthetic gene construct (SEQ ID NO: 1). The ligation mix was used fortransformation of E. coli TOP10 (Invitrogen) resulting in the yeastexpression construct pGBS414PPK-1. Subsequently, pGBK414PPK-1 wasrestricted with AscI and NotI. To create pGBS414PPK-3, an AscI/NotIrestriction fragment consisting of glycosomal fumarate reductase from T.brucei (FRDg) synthetic gene construct (SEQ ID NO: 2) was ligated intothe restricted pGBS414PPK-1 vector. The ligation mix was used fortransformation of E. coli TOP10 (Invitrogen) resulting in the yeastexpression construct pGBS414PPK-3.

The expression construct pGBS415FUM-3 was created after a BamHI/NotIrestriction of the S. cerevisiae expression vector pRS415 (Sirkoski R.S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligatingin this vector a BamHI/NotI restriction fragment consisting of thefumarase (origin Rhizopus oryzae) synthetic gene construct (SEQ ID NO:3). The ligation mix was used for transformation of E. coli TOP10(Invitrogen) resulting in the yeast expression construct pGBS415FUM-1.Subsequently, pGBK415FUM-1 was restricted with AscI and NotI. To createpGBS415FUM-3, an AscI/NotI restriction fragment consisting ofperoxisomal malate dehydrogenase from S. cerevisiae (MDH3) syntheticgene construct (SEQ ID NO: 4) was ligated into the restrictedpGBS415FUM-1 vector. The ligation mix was used for transformation of E.coli TOP10 (Invitrogen) resulting in the yeast expression constructpGBS415FUM-3.

The expression construct pGBS416MAE-1 was created after a BamHI/NotIrestriction of the S. cerevisiae expression vector pRS416 (Sirkoski R.S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligatingin this vector a BamHI/NotI restriction fragment consisting of theSchizosaccharomyces pombe malate transporter synthetic gene construct(SEQ ID NO: 5). The ligation mix was used for transformation of E. coliTOP10 (Invitrogen) resulting in the yeast expression constructpGBS416MAE-1.

1.1.2. Construction S. cerevisiae Strain

Plasmids pGBS414PPK-3, pGBS415FUM-3 and pGBS416MAE-1 (described under1.1.) were transformed by electroporation into S. cerevisiae strainRWB064 (MATA ura3-52 leu2-112 trp1-289 adh1::lox adh2::lox gpd1::Kanlox)to create strain SUC-200, overexpressing PCKa, MDH3, FUMR, FRDg andSpMAE1. All genes were codon pair optimized for expression in S.cerevisiae according to WO2008/000632.

1.2. Succinic Acid Production S. cerevisiae at Low pH and Oxygen LimitedConditions

The yeast strain SUC-200 (MATA ura3-52 leu2-112 trp1-289 adh1::loxadh2::lox gpd1::Kanlox, overexpressing PCKa, MDH3, FUMR, FRDg andSpMAE1), was cultivated in shake-flask (2×300 ml) for 3 days at 30° C.and 220 rpm. The medium was based on Verduyn (Verduyn et. al., 1992,Yeast 8, 501-517), but modifications in carbon and nitrogen source weremade as shown in Table 1.

TABLE 1 Preculture shake flask medium composition Compound Concentration(g/l) C₆H₁₂O₆•H₂O 20.0 (NH₂)₂CO 2.3 KH₂PO₄ 3.0 MgSO₄•7H₂O 0.5 1 1^(a)Vitamin solution Concentration Component Formula (g/kg) Biotin (D−)C₁₀H₁₆N₂O₃S 0.05 Ca D(+) panthothenate C₁₈H₃₂CaN₂O₁₀ 1.00 Nicotinic acidC₆H₅NO₂ 1.00 Myo-inositol C₆H₁₂O₆ 25.00 Thiamine chlorideC₁₂H₁₈Cl₂N₄OS•xH₂O 1.00 hydrochloride Pyridoxol hydrochloride C₈H₁₂ClNO₃1.00 p-aminobenzoic acid C₇H₇NO₂ 0.20 ^(b)Trace elements solutionFormula Concentration (g/kg) C₁₀H₁₄N₂Na₂O₈•2H₂O 15.00 (EDTA) ZnSO₄•7H₂O4.50 MnCl₂•2H₂O 0.84 CoCl₂•6H₂O 0.30 CuSO₄•5H₂O 0.30 Na₂MoO₄•2H₂O 0.40CaCl₂•2H₂O 4.50 FeSO₄•7H₂O 3.00 H₃BO₃ 1.00 KI 0.10Subsequently, the content of the shake-flasks was transferred to 10 Lfermenter (Startweight 6 kg), which contained the following medium:

TABLE 2 Main fermentation medium composition Concentration Raw materialFormula (g/l) Ammonium sulphate (NH₄)₂SO₄ 2.5 Potassium dihydrogenKH₂PO₄ 3.0 phosphate Magnesium sulphate MgSO₄•7H₂O 0.5 Trace elementsolution 1 Vitamin solution 1

The pH was controlled at 3.0 by addition of 6 N KOH. The temperature wascontrolled at 30° C. Glucose concentration was kept limited (<1 g/l) bycontrolling feed addition to the fermenter. Different oxygen uptakerates (OUR) were applied to the fermentation, which resulted in oxygenlimitation (FIG. 1).

0.33 vvm of total gasflow was applied, including 10% CO₂ to supplyenough CO₂ for efficient succinic acid production.

The results of different applied OUR's on the succinic acid productionare shown in FIG. 1. A minimal amount of aeration was required tosustain succinic acid production at a pH of 3. An OUR above 5 mmol/L/hresulted in lower succinic acid production.

During the cultivation of 90 hours, growth occurred to a typical biomassconcentration of 8 g dry weight/L. Consequently, the specific oxygenuptake rate (qO₂) decreased constantly during the fermentation. An OURof 10 mmol/L/h applied in one fermentation correlated with a qO₂decreasing from 10 to 1.25 mmol/g biomass dry weight/h and an OUR of 1mmol/L/h correlated with a qO₂ decreasing from 1 to 0.1 mmol/g biomassdry weight/h.

The invention claimed is:
 1. A process for the preparation of succinicacid said process comprising: a) a biomass formation phase comprisingcultivating a yeast under conditions sufficient to produce a biomass;and b) an acid production phase comprising fermenting the biomass in thepresence of a carbohydrate-containing substrate and low amounts ofoxygen at a pH value which is below the lowest pKa of succinic acid,wherein the oxygen is supplied at a specific oxygen uptake rate rangingbetween 8 to 0.2 mmol/g biomass dry weight/hour, wherein said acidproduction phase produces more than 10 g/L succinic acid duringcultivation for 90 hours.
 2. The process according to claim 1 whereinthe pH of the biomass formation phase is in the range of pH 2 to pH 7,and the pH of the acid production phase is in the range of pH 1.0 to pH5.5.
 3. The process according to claim 1, wherein the acid productionphase comprises fermenting the biomass under carbon-limited conditions.4. The process according to claim 1, wherein the acid production phaseis performed in the presence of an extra electron donor.
 5. The processaccording to claim 1, wherein the yeast is a Saccharomyces cerevisiae.6. The process according to claim 1, wherein the yeast is a geneticallymodified yeast.
 7. The process according to claim 6, wherein thegenetically modified yeast comprises a nucleotide sequence encoding aheterologous enzyme selected from the group consisting of a phosphoenolpyruvate carboxykinase, fumarate reductase and a fumarase.
 8. Theprocess of claim 7, wherein the heterologous enzyme is selected from thegroup consisting of an Actinobacillus phosphoenol pyruvatecarboxykinase, a Trypanosoma fumarate reductase and a Rhizopus fumarase.9. The process of claim 7, wherein the heterologous enzyme has at least95% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.